This application addresses the unmet need for high-throughput methods to identify active components in botanical dietary supplements and to determine their mechanisms of action through metabolomics studies. Ultrafiltration mass spectrometry (PUF-MS), which was invented in this laboratory, is more efficient than conventional bioassay-guided fractionation for the screening, characterization, isolation, and identification of pharmacologically significant ligands contained in botanical dietary supplements or other natural product mixtures. We propose to enhance the productivity of this technology 100-fold using a new ultrafast PUF-MS (UPUF-MS) approach and to develop new validated assays for the standardization of botanical dietary supplements based on UHPLC-MS-MS that are at least 10-fold faster than previously possible. We will incorporate UHPLC-MS into metabolomics studies of archived serum and urine samples from clinical trials of the effects of botanical dietary supplements black cohosh, red clover, hops, Prempro, and placebo on hot flashes in menopausal women. It is our hypothesis that this metabolomics approach will facilitate the discovery of biomarkers indicative of vasomotor symptoms such as hot flashes and night sweats. Then, these biomarkers may be used for the objective, quantitative determination of the effects of botanical dietary supplements on hot flashes instead of relying on subjective self-reporting of hot flashes by subjects in clinical trials. Overall, these studies will address te central hypothesis that botanical dietary supplements contain pharmacologically active compounds that can be identified using UPUF-MS, used for chemical standardization with UHPLC-MS-MS and that the in vivo effects of these active constituents can be detected using UHPLC-MS-MS in combination with metabolomics.